Versatile Site-Selective Protein Reaction Guided by WW Domain-Peptide Motif Interaction
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AbstractA short, flexible, and unstructured peptide tag that has versatile and facile use in protein labeling applications is highly desirable. Here, we report an 11-residue peptide tag with an internal cysteine (a W-tag, derived from a Comm PY peptide motif that is known to bind with Nedd4 WW3* domain) that can be installed at different regions of the target protein without compromising its covalent reactivity with the reactive label (a 35-residue synthetic Nedd4 WW3* domain derivative). This versatility is explained by the unique structural features of the reaction. NMR analysis reveals that both the W-tag peptide and reactive Nedd4 WW3* protein are sir unstructured before they encounter each other. The binding interaction of the two induces noticeable structural changes and promotes global folding. Consequently, the reactive cysteine residue at W-tag and the electrophilic chloroacetyl group at Nedd4 WW3* domain are positioned to be in close proximity, inducing an intermolecular covalent cross-linking. The covalent linkage in turn stabilizes the folding of the protein complex. This unique multistep mechanism renders this labeling reaction amenable to different sites of the proteins of interest: installation of the tag at N- and C-termini, in the flexible linker region, in the loop region, and the extracellular terminus of target proteins exhibited comparable reactivity. This work therefore represents the first proximity-induced cysteine reaction based on the unique binding features of WW domains that demonstrates unprecedented versatility.
All Author(s) ListLiu Miao, Ji Zeyang, Zhang Mingjie, Xia Jiang
Journal nameBioconjugate Chemistry
Volume Number28
Issue Number8
PublisherAmerican Chemical Society
Pages2199 - 2205
LanguagesEnglish-United States

Last updated on 2021-22-10 at 23:40